The splicing‐regulatory lncRNA NTRAS sustains vascular integrity

Abstract Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non‐coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type‐specific manner. By screening for endothelial‐enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA‐dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO‐1) pre‐mRNA. Deletion of the hnRNPL binding motif in mice (Ntras ∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α‐ isoform, which augments permeability of the endothelial monolayer. Ntras ∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.


2) Please explain in more detail in the manscript the refutation aspect to Lv et al. w.r.t transcriptional regulation vs. splicing. Include figs 1a-c 'for reviewers' (see also ref 2#13)
Following the editor's suggestion, we addressed the findings by Lv et al. and in this context integrated all above mentioned figures. The revised paragraph now reads: "In this context, a recently reported regulation of TJP1 total expression levels (and apoptosis-related proteins) by hnRNPL in epithelial cells (Lv et al., 2017) could not be observed for endothelial cells ( Figure EV3A, B). Likewise, NTRAS silencing in endothelial cells did not influence TJP1 total mRNA levels ( Figure EV3C). However, exon 20 splicing regulation by NTRAS was also evident in the epithelium ( Figure EV3D)."

3) Please ensure the manuscript includes a clear explanation of the '2 models' to explain TJP1 splicing regulation holistically (sequestration & recruitment cf. ref 1 #3).
Based on the limitation of our data to explain the synergistic splicing events (recruitment model), the recommendation to re-focus the revised manuscript on the mechanism of TJP1 splicing, and with reference to the pre-decision discussion with the editor, we removed data and figures emphasizing a putative recruitment model. This applies to original figures 2I and 2J. The confirmation of additional, NTRAS-hnRNPL co-regulated pre-mRNAs was moved to Appendix figure 1B-F and is now cited in the revised discussion which deals with the interesting aspects that 1. NTRAS-hnRNPL might regulate additional transcripts beyond TJP1 and 2. This might be achieved by other mechanisms than hnRNPL sequestration. The revised paragraph comprises lines 275 to 291.

4) Include control Fig 3A-D 'for reviewers' that addresses ref. 1#4 as well as ref 2#9.
To underline the specificity of our identified NTRAS-hnRNPL axis, we followed the editor's advice and included an additional lncRNA control for the assessment of genome-wide splicing regulation. This new data is shown in Figure EV2J of the revised manuscript. In this context, we also demonstrated the specificity of hnRNPL on TJP1 exon 20 inclusion by silencing of the splicing factor hnRNPU which failed to reproduce the outcome of hnRNPL silencing. This supporting data is now shown as new Figures EV2K, L. The combined section now reads: "Of note, silencing of an unrelated control lncRNA and hnRNPU, a heterogeneous nuclear ribonucleoprotein not associated with NTRAS, failed to regulate TJP1 exon 20 inclusion rates ( Figure EV2J-L)." Finally, we included data demonstrating unchanged endothelial permeability upon silencing of hnRNPU, see Figure EV3L of the revised manuscript. The paragraph addressing this new data reads: "In contrast, silencing of hnRNPL ( Figure EV3J) specifically augmented barrier function (Fig 3H and Figure EV3K), whereas silencing of the non-specific splicing factor hnRNPU had no effect ( Figure  EV3L)."

5) It is Ok to remove the HIF1 expression regulation data (ref 2#1), but it would appear reasonable to include the induction of hnRNP-NTRAS interaction data 'fig 4D for reviewers' (cf. ref 2 #7 & #12)
As suggested by the editor, we removed the HIF1 data from the revised manuscript and included data on the augmented interaction between NTRAS and hnRNPL following hypoxia. This new data is shown in Figure EV2F of the revised manuscript. As requested by the editor, we included the sucrose density gradient ultracentrifugation showing the overlapping distribution of NTRAS and hnRNPL as new Figure EV2D of the revised manuscript. The rearranged paragraph reads: "Given that hnRNPL is a highly expressed protein (Beck et al., 2011) whereas NTRAS is rather a low abundant lncRNA, we questioned the stoichiometry of both factors. To this end, we deployed density gradient ultracentrifugation ( Figure EV2D) revealing that the majority of hnRNPL (~ 79 %) is not bound to NTRAS. However, a major fraction of NTRAS co-sediments with hnRNPL, supporting the supposed interaction of both factors. This result is in line with the circumstance that hnRNPL is engaged in a multitude of different RNA-binding processes, whereas the association with NTRAS might be involved in fine tuning a specific subset of hnRNPL-mediated processes. In addition, in silico analysis of the NTRAS sequence revealed several CA-rich hnRNPL binding motifs and strikingly a prominent bona fide hnRNPL binding site in the form of a CA16 repeat sequence proximal to the 3' splice site of the predominantly retained intron 2 ( Figure EV2E). Therefore, it might be reasonably assumed that the presence of multiple hnRNPL binding motifs within NTRAS will compensate for the unfavorable stoichiometry between both factors. Finally, RNA immunoprecipitation ( Fig 2D) and RNA affinity selection followed by western blotting ( Figure EV2F) unequivocally validated the interaction between NTRAS and hnRNPL. Furthermore, such interaction was enhanced under hypoxia-mediated NTRAS upregulation, corroborating the aforementioned data ( Figure EV2F). In summary, our results suggest that NTRAS exists as a constituent of an hnRNPLcontaining ribonucleoprotein complex in the nucleus.

8) Include the explanation to ref 2#19 in the manuscript.
We followed the editor's advice and clarified the usage of the two-exon mini-gene construct for our in vitro splicing assays. The revised passage now reads: "First, we assessed the in vitro splicing efficiency of a TJP1 minigene construct upon NTRAS depletion in splicing competent nuclear extract. Since the in vitro transcription of an exon 19-20-21 TJP1 minigene proved to be inefficient, we deployed a previously described construct, comprising the constitutive exon 19, intron 19 (which contains the hnRNPL binding motifs), and the alternative exon 20 (Fig. 3A) (Heiner et al., 2010). RNase H-mediated NTRAS degradation in nuclear extracts prior to splicing ( Figure EV3E) significantly diminished the splicing efficiency of the TJP1 exon 19-20 minigene (Fig 3B). Strikingly, this effect could be rescued by the addition of an in vitro transcribed NTRAS fragment, harboring the CA 16 dinucleotide repeat, prior to splicing (Fig 3B)." With reference to the pre-decision discussion od the arbitrating referee's comments, please proceed as suggested to: (i) assess TJP1 pre-mRNA splicing in vitro upon RNaseH-mediated degradation of NTRAS including the control.
We successfully rescued the impaired splicing efficiency of our TJP1 mini-gene, induced by RNaseHmediated degradation of NTRAS, by add-back of an NTRAS fragment harboring the major hnRNPL binding motif. This new data demonstrates the binding competition between hnRNPL, NTRAS and the TJP1 pre-mRNA and is shown in the revised manuscript as new Figure 3B, thereby replacing the previously shown in vitro splicing data. The corresponding section reads: "RNase H-mediated NTRAS degradation in nuclear extracts prior to splicing ( Figure EV3E) significantly diminished the splicing efficiency of the TJP1 exon 19-20 minigene ( Fig 3B). Strikingly, this effect could be rescued by the addition of an in vitro transcribed NTRAS fragment, harboring the CA 16 dinucleotide repeat, prior to splicing (Fig 3B)." (ii) tone down generalized mechanism of action for the splicing regulatory function of NTRAS-hnRNPL.
In accordance with our response to comment 3), we re-focused our revised manuscript on the molecular mechanism of NTRAS-hnRNPL regulating TJP1 exon 20 usage and eventually endothelial permeability.
To this end, we removed most of the data addressing a general splicing regulatory mechanism, specifically the original figures 2I and 2J. However, to indicate that our observed splicing regulatory processes are not strictly limited to TJP1 exon 20, we moved the additionally validated NTRAS-hnRNPL splice substrates to Appendix figure 1B-F and chose to address the notion that both factors might be part of a more complex splicing network in the discussion; see lines 275 to 291.

Please include:
1) A data availability section providing access to data deposited in public databases is missing. If you have not deposited any data, please add a sentence to the data availability section that explains that.
A data availability section is provided in the revised manuscript and RNA sequencing and mass spectrometry data were deposited in a publicly available repository. RNA sequencing data can be accessed via the identifier E-MTAB-11311, and the mass spectrometry data via PXD030620.

In particular, the referee noted the impact of NTRAS depletion is not convincing and suggested the rescue with the endogenous gene, not a minigene.
We thank the reviewer for this suggestion, however, want to clarify that based on the transcript length of TJP1, an in vitro splicing assay using the full-length TJP1 pre-mRNA is technically not feasible. Minigenes, in turn, proved to be valuable tools to specifically assess splicing patterns of interest. Of note, the TJP1 minigene deployed by us has been successfully used by others (e.g. Heiner et al., 2010) to identify and analyse the splicing repressive function of hnRNPL on TJP1 exon 20.
Finally, we would like to point out that we have already demonstrated the effect of the native endogenous RNA on splicing by overexpression of NTRAS in Fig. 3F of the manuscript.

The referee is not convinced by the statistical significance of the new data: 'Appendix figure panel C cannot be a two-star significance. Or Appendix F. Or Fig. 3B as mentioned before, or 3D, 4G, etc... if data points overlap, how come it is significant?'
Please note that we performed the appropriate statistical analysis for all figures with n ≥ 3 biological replicates. The results confirm p-values below 0.05 (see screen shots provided in Figure 1 for the reviewer), which is accepted as gold standard for concluding statistically significant differences. In addition, we are happy to provide the source data along with our submission, allowing the reviewer to confirm our analysis. Of note, columns with overlapping data points can be significantly different, if the statistical test used compares the means of the individual sample groups, which is e.g. the case for Student's t-tests. Finally, we want to state that the results shown in Appendix Figure S1A-F were primarily included to support / verify the n = 2 RNA sequencing results of Fig. 2H, where no statistical analysis is possible.
3. The referee also raises a potential discrepancies: re. Appendix Figure 1: 'KD of NTRAS represents an overexpression of hnRNPL. How come the KD of the lncRNA impacts splicing in the same direction as KD of the splicing factor? How come TJP1 ex20 changes are not significant.
Response to the first part of the reviewer's comment: "KD of NTRAS represents an overexpression of hnRNPL." We want to emphasize that we never showed, assumed, or suggested that NTRAS regulates hnRNPL expression positively or negatively. What the reviewer might have misunderstood in Appendix Figure S1A is that NTRAS silencing (blue column; LNA NTRAS) represses TJP1 exon 20 inclusion (please see axis-title) compared to the control condition (light grey; LNA Ctrl). On the other hand, hnRNPL silencing (pink column; si hnRNPL) enhances exon 20 inclusion compared to the control condition (dark grey; si Ctrl).
Response to the second part of the reviewers comment: "How come the KD of the lncRNA impacts splicing in the same direction as KD of the splicing factor?" Indeed, we report some examples in which NTRAS knock down impacts splicing of pre-mRNAs in the same direction as knockdown of hnRNPL. However, we do not claim that these events are necessarily causally linked and the main intention of showing the data provided in the Appendix was to validate the RNA sequencing data. Of note, based on our recent correspondence with EMBO Reports and the agreement to focus on TJP1 splicing while toning down statements addressing transcriptome-wide splicing-regulatory mechanisms of NTRAS-hnRNPL, we decided to remove the complex transcriptome data from the revised manuscript. We believe that this greatly enhances the accessibility of our manuscript and while we agree that a detailed transcriptome-wide mechanistic analysis is thrilling, we consider this to be beyond the scope of our actual manuscript. Nevertheless, we hope that our sequencing data might be the starting point for follow-up studies, specifically dedicated to this very interesting mechanistic detail.
Response to the third statement: "How come TJP1 ex20 changes are not significant." We have sequenced two biological replicates per condition. This study design precludes a statistical analysis but was meant to be hypothesis generating. Please note that the data on TJP1 exon 20 usage were subsequently validated by various experiments. E.g. We apologize for any misunderstandings and want to clarify that Fig. EV2J shows the overall differential splicing changes upon silencing of an unrelated lncRNA (lincflow2). In this analysis, only 3 skipped exons were affected compared to 131 exon skipping events upon NTRAS silencing (Fig. 2G).
The 6 exons analysed in the appendix, as the figure legend indicates, are examples of these NTRAS-regulated alternative splicing events.

5.
Overall, I am not sure the model is right. I believe NTRAS sequesters hnRNPL. That this has a biological impact, I am not that sure. The data is not strong enough and the whole model just stands for one exon, which I do not understand why if it is a sequestration mechanism...where the specificity comes from?
Regarding the reviewer's concern, we specifically and exclusively propose a sequestration model for splicing of TJP1 exon 20. This model is based on ample mechanistic evidence and we eventually demonstrate the biological impact of NTRAS-hnRNPL-mediated regulation of TJP1 splicing.
A short summary of the key data supporting our conclusions is listed below and shown in Figures 2-5

for the reviewer:
For the sequestration of hnRNPL by NTRAS, please see Figures 2 and 3 for the reviewer: Fig. 2A and B for the reviewer show the presence of bona fide hnRNPL binding motifs in human and mouse NTRAS transcripts along with numerous lower-ranking binding sites.